Methods and tissue culture media for inducing somatic embryogenesis, agrobacterium-mediated transformation and efficient regeneration of cacao plants

ABSTRACT

The present invention relates to improved methods of (i) inducing somatic embryogenesis from cacao tissue explants and (ii) regenerating cacao plants from somatic embryos. The invention further relates to cacao somatic embryos and plants obtained according to the methods of the invention. Novel tissue culture media adapted for use in the above-identified methods are also within the scope of the invention. The novel media of the invention include primary callus growth medium, secondary callus growth medium, embryo development medium, primary embryo conversion medium, secondary embryo conversion medium and plant regeneration medium.

[0001] The present invention claims priority under 35 U.S.C. §119 ofprovisional applications Nos. 60/051,147 filed Jun. 27, 1997; 60/069,704filed Dec. 16, 1997; and 60/051,133 filed Jun. 27, 1997, the disclosuresof which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to an improved method of inducingsomatic embryogenesis and regenerating cacao plants from cacao tissue.The invention also relates to a method for transforming cacao usingAgrobacterium-mediated transformation to introduce novel geneticmaterial into cacao plant DNA and regenerating transgenic plants fromtransformed callus and somatic embryos. The invention further relates tonovel culture media such as primary callus growth medium, secondarycallus growth medium, embryo development medium, primary embryoconversion medium, secondary embryo conversion medium, and plantregeneration medium which media are adapted for use in the methods ofthe invention.

BACKGROUND OF THE INVENTION

[0003] Cacao (Theobroma cacao L.) is the second most importantagricultural commodity in the international trade market for thetropical regions. Cacao powder and cacao butter, which are obtained fromthe processed cacao beans, are the most important ingredients inchocolate and confectionery products, and are also important additivesin pharmaceutical and cosmetic products. Due to increasing demands forcacao-related products, there is an ever greater need for cacao treeswith higher levels of productivity and improved cacao bean quality.Cacao trees have a high degree of genetic heterozygosity. A typicalcacao planting contains a large population of trees grown fromgenetically different seeds, and relatively few trees produceexceptional yields, and about one third of the trees produce belowaverage yields.

[0004] Since the majority of cacao commercially cultivated today isderived from a few varieties collected 50-60 years ago and has a narrowgenetic base, cacao remains extremely vulnerable to diseases and otherabiotic stresses. Up to 30% of the world cacao crop production is losteach year due to fungal and viral diseases and to attack by variousinsect pests (Wood and Lass 1987, Cocoa, 4th edition. Longman Sci & Techand John Wiley & Sons, NY). In 1995, almost 50% of the total cacao beanproduction in Brazil was lost due to the witch's broom disease pathogen,and this raised concerns within the cacao industry, over the need forproduction of disease-resistant varieties. Continued improvement incacao production, through the development and utilization of superiorgenotypes with desirable yield and bean quality characteristics,resistance to diseases and insect pests, and tolerance to drought andcold, via conventional breeding methods and biotechnology approaches,remains a great challenge. In this regard, the development of a reliabletransformation system for cacao has become critical for the successfulutilization of biotechnology for cacao tree improvement. Furthermore, itis desirable to be able to propagate vegetativelly higher-yielding treesto ensure uniform high yields.

[0005] During the past thirty years, attempts were made to usevegetative cloning of superior genotypes or selected trees producedthrough breeding as a means to increase the overall yield, quality, andagronomic performance of cacao. However, in spite of a great deal ofeffort over a number of years to devise improved methods for vegetativepropagation, cacao trees are currently commercially reproduced only viacuttings. There are a number of disadvantages associated with thepropagation of cacao plants via the rooting or grafting of plagiotropiccuttings. For example, this mode of propagation is expensive and laborintensive, propagation rates are low, there is a wide range of variationin the performance of individual cuttings, an undesirable bush-likegrowth pattern may occur, and there is a high degree of susceptibilityto wind damage due to the lack of a taproot system. Thus, there is agreat need in the art of cocoa cultivation for an efficient clonalpropagation method that could provide plants agronomically similar toseed-derived plants.

[0006] Considerable effort was made to develop tissue culture-basedpropagation methods. However, cacao has proven to be notoriouslyrecalcitrant to in vitro propagation (Flynn et al., (1990) Plant Tissueand Organ Cult. 20:111-117; Passey and Jones, (1983) J. Hort. Sci58:589-592; Orchard et al., (1979) Physiol. Plant. 47:207-210).

[0007] Plant regeneration through somatic embryogenesis provides analternative approach for clonal propagation of cacao. Somaticembryogenesis is the process by which somatic cells undergo bipolardevelopment to give rise to whole plants by means of the development ofadventitious embryos that occur without the fusion of gametes. Plantsderived from somatic embryos are genetically identical to their parentaldonor cells, and have a taproot system and an orthotropic growth patternsimilar to that of seed-derived plants.

[0008] Certain studies on somatic embryogenesis and plant regenerationof cacao have been performed. For example, Janick et al. (U.S. Pat. Nos.4,204,366; 4,291,498; 4,301,619 and 4,545,147) and Sondahl et al. (U.S.Pat. No. 5,312,801) studied the possibility of inducing embryogenesisand regenerating plants of cacao.

[0009] Janick discloses a method for producing somatic embryosexclusively from immature zygotic embryo tissues of cacao using MS-basedmedium and increased (3-fold) CO₂ concentration. In that procedure, theconversion or germination of somatic embryos into seedlings or plantletswas problematic and mature plantlets were not obtained. (See, e.g. Wangand Janick, (1984) Hort. Sci. 19:839-841). Furthermore, as pointed outby the same inventors in a subsequent publication (Figueira and Janick,(1993) Acta Hortic. 336:231-236), somatic embryos derived from immaturezygotic embryos have limited value for commercial propagation, becausecacao seeds are produced mainly through open pollination and the zygoticembryos used as a starting material are untested genotypes, i.e., thezygotic tissues are not genetically identical.

[0010] Sondahl developed a method for inducing somatic embryogenesis andplant production which uses a non-zygotic somatic tissues obtained frommature cacao plants as a starting material. The Sondahl procedure usesan MS-based culture medium and high sugar content. The procedureinvolves the following steps: (i) inducing a friable embryogenic callusfrom non-somatic tissues in a callus induction medium; (ii) recoveringimmature embryos from the friable embryogenic callus in a liquid culturemedium; (iii) producing first stage somatic embryos in a regenerationmedium; (iv) developing second stage somatic embryos in adifferentiation medium with a high osmotic potential (80-120 g/lsucrose); and (v) germination of mature somatic embryos in plantregeneration medium. Sondhal uses ABA and GA hormones for embryoinduction. In this procedure, regeneration of cacao plantlets dependsprimarily on the secondary somatic embryos induced from primary embryossubjected to an extended culture period. Up to 8 different types ofculture media, and multiple growth regulators such as cytokinins(including zeatin, kinetin, 6-BA and 2-iP), auxins (including NAA andIAA), gibberellic acid, and abscisic acid were required.

[0011] The development of a procedure for inducing somatic embryogenesisin non-zygotic tissues as described by Sondahl did not eliminate theproblem associated with in vitro propagation of cacao. The procedurecould not be applied to all somatic tissues of cacao. Only two types oftissue explants, nucellus (the inner layer of an ovule) and young flowerbud petals, were responsive to the established culture conditions andwere capable of producing somatic embryos. This is a significantdisadvantage since nucellus tissue can only be obtained from young cacaofruits and the availability of young fruits is often limited. Cacaoplants generally have a low number of fruits because the majority ofyoung fruits tend to abort during development.

[0012] More importantly, the Sondahl procedure resulted in a very lowfrequency of somatic embryogenesis and plant regeneration. For example,according to examples cited in the patent description, only 8 cacaoplants were successfully established in the soil from 30,160 culturednucellus explants that generated a total of 948 primary somatic embryo,and only 7 plants were produced from 27,721 cultured petal explants thatproduced a total of 167 primary embryos (U.S. Pat. No. 5,312,801).Additionally, the Sondahl procedure was tested using only two cacaogenotypes (EET-162 and UF-667). A recent study by a French group usingthe Sondhal method demonstrated that only 5 among 25 tested cacaogenotypes were capable of producing somatic embryos, while the restremained non-responsive (Lopez-Baez et al., (1993) CRAS, Paris316:579-584). Thus, the utilization of the procedures known in the arthas never been attempted commercially. The low frequency ofembryogenesis and plant production and the inability to produce somaticembryos from the majority of cacao genotypes have precluded thepractical use of the Janick and Sondahl methods. Accordingly, thereremains a need in the art for the development of an efficient method forthe regeneration of cacao plants.

[0013] Applicants have now developed effective procedures for thestimulation of somatic embryogenesis and plant regeneration from somatictissues of cacao. The present procedure has significant advantages overthe Janick and Sondhal procedures. The procedure of the presentinvention uses novel culture media that are not based on the MS basalmedium. In fact. Applicants have shown that the MS medium is toxic tocacao cells, which may explain the low efficiency of the Janick andSondhal procedures. Furthermore, the method of the invention does notrequire high CO₂ levels (as described by Janick) or high osmoticum(80-120 g/l sucrose as described by Sondhal). The embryo conversionmedium of the invention is effective without high sucrose levels andwithout growth hormones. In contrast, the Sondhal procedure requireshigh osmoticum and hormones. Accordingly, Applicants have nowsurprisingly discovered a novel procedure and novel culture media thatare more effective and simpler to use than those described by the priorart.

[0014] Genetic transformation of plant cells offers a unique method tomodify the plant genetic milieu and thus expedite the introduction ofvaluable agronomic traits into existing genotypes. Two major approaches,biolistics (gene gun) and Agrobacterium tumefaciens-mediated genetransfer, have been developed for gene introduction in many plantspecies. The biolistics approach involves the introduction of DNA thatis carried on metal particles which are accelerated by a high velocityforce into target plant cells. Agrobacterium-mediated transformation isaccomplished by utilizing the natural DNA delivery capabilities of theA. tumefaciens bacterium.

[0015] Over the years, attempts have been made by a number of researchgroups to develop workable transformation protocols for cacao using bothof the above-mentioned methods. However, successful transformation ofcacao using the biolistics approach has not been demonstrated. Thereports of Purdy and Dickstein (Plant Disease 73: 638-639; 1989) andSain et al. (Plant Cell Tiss Org Cult 37:243-251; 1994) provided thefirst evidence that a wild type A. tumefaciens strain is capable oftransferring and integrating the T-DNA into the cacao genome. However,only non-regenerable tumorous callus tissue was obtained, and the use ofnon-tumorigenic strains of A. tumefaciens that had been modified tocontain a disarmed Ti plasmid, failed to infect cacao cells.

[0016] Applicants have now developed a protocol for transformation ofthe somatic embryos and the production of transgenic cacao embryos andplants using non-tumorigenic strains of A. tumefaciens. Applicants arethe first to obtain transformed cacao plants.

SUMMARY OF THE INVENTION

[0017] The present invention relates to an improved method ofregenerating cacao plants by inducting somatic embryogenesis from cacaotissue explants and regeneration of cacao plants from somatic embryosusing novel culture media. The culture media of the invention includeprimary callus growth medium, secondary callus growth medium, embryodevelopment medium, primary embryo conversion medium, secondary embryoconversion medium and plant regeneration medium. The invention alsorelates to a method for transforming cacao tissues with A. tumefaciensand producing transgenic cacao plants.

[0018] Accordingly, in one aspect, the invention provides for a methodof obtaining somatic embryos by culturing cacao tissue explants.

[0019] In another aspect, the invention relates to a method ofregenerating cacao plantlets and mature cacao plants from cacao somaticembryos.

[0020] In yet another aspect, the invention relates to culture media(solid, semi-solid and liquid) adapted for induction of somaticembryogenesis and regeneration of cacao plants.

[0021] In yet another aspect, the invention relates to a method ofinducing an Agrobacterium mediated transformation of cacao andregeneration of transgenic plants.

[0022] In a further aspect, the invention relates to somatic embryos andcacao plants obtained according to the methods of the present invention.

DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1A represents a schematic outline of cacao plant regenerationin tissue culture according to one embodiment of the invention.

[0024]FIG. 1B represents a schematic outline of cacao plant regenerationin tissue culture according to one embodiment of the invention.

[0025]FIG. 2 shows certain steps in the process of culturing staminodeexplants inducing mature embryos and regenerating plantlets. FIG. 2(a)is a photograph of cultured staminode explants. FIG. 2(b) is aphotograph of embryonic callus induced from the entire staminodeexplant. FIGS. 2(c), (d) and (e) are photographs of somatic embryos atvarious stages of development (globular, heart and torpedo-shapedembryos). FIGS. 2(f) and (g) are photographs of mature embryos andconverted plantlets respectively. FIG. 2(h) is a photograph of somaticembryo-derived cacao plants grown in the greenhouse.

[0026]FIG. 3 is a graph representing the effects of TDZ concentration onsomatic embryogenesis from staminode explants for five cacao genotypesusing the culture procedures according to one embodiment of theinvention. The percentile of responsive staminodes at each TDZconcentration is shown above the bars.

[0027]FIG. 4 is a graph representing the effects of TDZ concentration onsomatic embryo production from staminode explants for five cacaogenotypes using the culture procedures according to one embodiment ofthe invention. The average number of somatic embryos per responsivestaminode at each TDZ concentration is shown above the bars.

[0028]FIG. 5 is a graph representing the percentile of staminodesresponsive to somatic embryo formation from staminode explants of 19tested cacao genotypes using the culture procedures according to oneembodiment of the invention.

[0029]FIG. 6 is a graph representing the average number of somaticembryos produced from responsive staminode explants of 19 tested cacaogenotypes using the culture procedures according to the invention.

[0030]FIG. 7 is a graph showing the effect of the concentration of6-benzylaminopurine in the secondary callus growth medium on thefrequency of somatic embryo production from staminodes.

[0031]FIG. 8 is a graph showing the effect of the concentration of6-benzylaminopurine in the secondary callus growth medium on the averagenumber of somatic embryos produced per responsive staminode explant.

[0032]FIG. 9 is a schematic outline of the procedure forAgrobacterium-mediated transformation of cacao according to one aspectof the invention.

[0033]FIG. 10 is a photograph representing the expression of GFP inputative transgenic somatic embryos of cacao afterAgrobacterium-mediated transformation.

[0034]FIG. 11 depicts the T-DNA region of the binary vector pDM96.0501.RB and LB represent the right and left border of T-DNA, 35S-p and 25S-trepresent the CaMV35S promoter and terminator, SGFP represents the greenfluorescent protein gene, GUS represents the beta-glucuronidase gene,NPTII represents the neomycin phosphotransferase genes, and tml3′represents the tumor morphology large gene terminator.

[0035]FIG. 12 is a graph showing the effect of developmental stages ofcacao somatic embryos and gene promoters on transient GFP expressionafter Agrobacterium-mediated transformation.

[0036]FIG. 13 is a graph representing transient GFP expression insomatic embryos of cacao genotype Sca-6 after Agrobacterim-mediatedtransformation.

DETAILED DESCRIPTION OF THE INVENTION

[0037] All patents, patent applications and publications cited hereinare hereby incorporated by reference. In case of inconsistencies thepresent disclosure governs.

[0038] The present invention relates to improved methods of (i) inducingsomatic embryogenesis from cacao tissue explants, (ii) regeneratingcacao plants from somatic embryos and (iii) transforming cacao tissuesand regenerating transgenic plants. The invention further relates tocacao somatic embryos and plants obtained according to the methods ofthe invention. Novel tissue culture media adapted for use in theabove-identified methods are also within the scope of the invention. Thenovel media of the invention include primary callus growth medium,secondary callus growth medium, embryo development medium, primaryembryo conversion medium, secondary embryo conversion medium and plantregeneration medium.

Method of Inducing Somatic Embryos

[0039] According to one embodiment of the invention, a method forinducing somatic embryos from cacao tissue explants is provided. Themethod generally includes the steps of obtaining cacao tissue explants,culturing explants to obtain callus and inducing somatic embryos in thecallus.

[0040] Any cacao tissue may be used as a source of explants. Forexample, cotyledons from seeds, young leaf tissue, root tissues, partsof stems including nodal explants, and tissues from primary somaticembryos such as the root axis may be used. Generally, young tissues area preferred source of cacao explants. In one preferred embodiment floraltissue explant, including staminodes (needlelike tissue fused withfilament at the base of stamens) and petal base (cup-shaped pouch) areused. Cacao trees continuously produce flowers (year-round) and thusprovide an unlimited source of floral explants. The advantage of thepresent invention is that it is effective on non-zygotic tissues.However, zygotic tissues may also be used.

[0041] Cacao explants are then placed on a primary callus growth mediumto induce callus, which may be compact or friable. In one embodiment ofthe invention a rapidly growing compact callus is induced.

[0042] The “primary callus growth culture medium” (PCG) has the propertyof inducing callus growth and is a basal tissue culture mediumcharacterized by a high content of the following ions: Ca, SO₄, Mg, PO₄,a low content of Cl and a higher ratio of NO₃/NH₄. For example, in oneembodiment, PCG is characterized by a high content of calcium nitrateand potassium sulfate and a low content of calcium chloride and havingthe property of inducing callus growth. The meaning of the term “basal”is understood to mean a medium containing essential macro- andmicronutrients. The meaning of the terms “high content” and “lowcontent” is understood to be in reference to both Murashige and Skoog(MS) medium (Murashige and Skoog (1962) Physiol. Plant 15:473-497) andWPM medium. The MS medium has the following concentration of these ions(in mM):43.4 NO₃; 21.0 NH₄; 3 Ca; 1.6 SO₄; 1.5 Mg; 1.25 PO₄ and 3 Cl.Thus, the “high content” of the PCG medium for Ca ion, for example, isabove 3 mM.

[0043] For example, the concentration of calcium nitrate may be fromabout 386.0 to about 2000.0 mg/l, preferably from about 800.0 to 1500.0mg/l, most preferably about 1367.0 mg/l. The concentration of potassiumsulfate may be from about 500.0 to about 2000.0 mg/l, preferably fromabout 990.0 to about 1800.0 mg/l, most preferably about 1559.0 mg/l. Theconcentration of calcium chloride may be from about 72.5 mg/l to about150.0 mg/l, preferably from about 72.5 to about 112.5 mg/l, mostpreferably 112.5 mg/l. The primary growth medium may contain othercomponents which may be in the ranges shown in Table 1. TABLE 1 DKW(mg/l) PCG (mg/l) Ammonium Nitrate 1416.0 400-2000 Boric Acid 4.80.3-10.6 Calcium Chloride Anhydrous 112.5 56-453 Calcium Nitrate 1367.0386-2000 Cupric Sulfate-5H₂O 0.25 0.006-0.5   Na₂-EDTA 45.4 10-75 Ferrous Sulfate-7H₂O 33.8 13-50  Magnesium Sulfate 361.49 17-903Manganese Sulfate-H₂O 33.5 0.76-50    Molybdic Acid (sodium salt)-2H₂O0.39 0.0025-1.25   Nickel Sulfate-6H₂O 0.005   0-0.01 PotassiumPhosphate Monobasic 265.0 68-400 Potassium Sulfate 1559.0 500-2000 ZincNitrate-6H₂O 17.0  5-30

[0044] Preferably, the primary callus growth medium comprises a DKWbasal medium prepared according to Driver and Kuniyuki, Hortsci.19:507-509 (1984). The composition of the DKW basal medium is shown inTable 1. DKW medium contains the following concentration of the ions (inmM): 34.3 NO₃; 17.7 NH₄; 8.3 Ca; 12 SO₄; 3 Mg; 1.9 PO₄ and 1 Cl.

[0045] The primary callus growth medium may be supplemented with atleast two growth regulators such as, for example, a cytokinin, an auxinor a combination thereof. Preferably, thidiazuron (TDZ) is used but anycytokinin may be used. Most preferably, a combination of TDZ and 2,4dichlorophenoxyacetic acid (2,4-D) are used. TDZ may be in theconcentration of from about 0.1 μg/l to about 100 μg/l, preferably about2.5 μg/l to about 50 μg/l, and most preferably from about 5 μg/l toabout 10 μg/l. 2,4-D may be in the concentration from about 0.5 mg/l toabout 5 mg/l, from about 0.8 mg/l to about 2 mg/l, and most preferablyabout 1.5 mg/l to about 2.0 mg/l.

[0046] The primary callus growth medium contains glucose or sucrose as acarbon source. Preferably, glucose is used. The concentration of thecarbon source may be as generally used in the art and for example, fromabout 15 g/l to about 45 g/l, preferably about 20 g/l.

[0047] MS medium and McCown's WPM woody plant basal medium are notsuitable for use as a basal media for the primary callus growth becausethe experiments have shown that they induce necrosis and reduce growth.

[0048] The explants are generally cultured on the primary callus growthmedium for about 10 to about 30 days, and preferably about 14 days. Theexplants are cultured at temperatures generally known in the art asuseful for callus growth, and for example, at 25±5° C.

[0049] The callus produced upon culturing cacao explants on primarycallus growth medium is subcultured on a secondary callus growth medium.The “secondary callus growth medium” (SCG) is a basal culture mediumcharacterized by a low salt concentration. The secondary callus growthmedium has the property of “conditioning” the callus growth, i.e.,limiting the callus growth and stimulating embryogenic homeostaticgrowth and bipolar callus cell development. Subculturing callus on SCGmedium enhances subsequent embryo differentiation from callus cells. Theelimination of this subculture step may result in excessive callusproliferation and a dramatic reduction in the frequency of somaticembryogenesis.

[0050] The secondary callus growth medium may contain from about 50% toabout 25% salt concentration of culture media generally known in theart. Any media known in the art (e.g. DKW, MS) diluted as describedabove may be used. Preferably, a low salt WPM basal medium described byLloyd and McCown, Int. Plant Prog. Soc. Proc. 30:421-427 (1981) may beused. The components and concentration of WPM medium as well as theranges of compounds that may be used are shown in Table 2. TABLE 2 WPM(mg/l) SCG (mg/l) Ammonium Nitrate 400.0 400-2000 Boric Acid 6.20.3-10.6 Calcium Chloride Anhydrous 72.5 56-453 Calcium Nitrate 386.0386-2000 Cupric Sulfate-5H₂O 0.25 0.006-0.5   Na₂-EDTA 37.3 10-75 Ferrous Sulfate-7H₂O 27.8 13-55  Magnesium Sulfate 180.7 17-903Manganese Sulfate-H₂O 22.3 0.76-33    Molybdic Acid (sodium salt)-2H₂O0.25 0.0025-1.25   Potassium Phosphate Monobasic 170.0 68-400 PotassiumSulfate 990.0 500-2000 Zinc Sulfate-7H₂O 8.6 0.2-43  

[0051] The secondary callus growth medium is supplemented with at leastone growth regulator. At least one cytokinin, such as for examplekinetin or 6-benzyl adenine (6-BA), and in some embodiments at least oneauxin, such as for example, 2,4-D may be used. Kinetin may be used inthe concentration of from about 0.01 mg/l to about 1 mg/l, preferablyabout 0.1 mg/l to about 0.5 mg/l, and most preferably from about 0.2mg/l to about 0.3 mg/l. 6-BA may be used in the concentration from about0.01 mg/l to about 1 mg/l, from about 0.05 mg/l to about 1 mg/l, andmost preferably from about 0.05 mg/l to about 0.2 mg/l. 2,4-D may beused in concentration from 0.9 to 3 mg/l, most preferably from 0.9 to2.0 mg/l.

[0052] In one embodiment, the secondary callus culture medium maycontain a combination of coconut water (in the place of 6-BA) andkinetin as growth regulators. Kinetin may be used in the concentrationsdescribed above, while coconut water may be used in the concentrationfrom about 25.0 to about 200.0 ml/l, preferably from about 25.0 to about150.0 ml/l and most preferably from about 50.0 to 100.0 ml/l.

[0053] TDZ is not used in the secondary callus culture medium.

[0054] The secondary callus growth medium contains glucose or sucrose asa carbon source. Preferably, glucose is used. The concentration of thecarbon source may be as generally used in the art and for example, fromabout 10 to 40 g/l, preferably from 20 to 30 g/l.

[0055] The callus is generally cultured on the secondary callus growthmedium for about 10 to about 30 days, and preferably about 14 days attemperatures generally known in the art and for example 25±5° C.

[0056] The callus conditioned on the secondary callus growth medium isthen transferred onto the embryo development medium which has theproperty of inducing formation of differentiated embryos. The “embryodevelopment medium” is a basal culture medium having the samecomposition as the basal medium of the primary callus growth medium. Forexample, the basal medium may be DKW basal medium. However, the embryodevelopment medium is not supplemented with growth hormones. The mediumcontains a carbon source and may contain sucrose from about 10 to 60g/l, preferably about 30 g/l.

[0057] The embryogenic callus is generally cultured on the embryodevelopment medium for about 14 to about 60 days, and preferably about30 days. During this period, a number of mature somatic embryos arevisible in the callus. These embryos may then be used to regeneratecacao plants or as a source of cacao tissue for production of secondaryembryos by following the procedure outlined above.

[0058] The above described steps of the method for inducing somaticembryos in cacao explants may be carried out in the dark or under thelight. Preferably, the steps are carried out in the dark.

Method of Regenerating Cacao Plantlets

[0059] The present invention further relates to a method forregenerating cacao plantlets and mature plants from cacao somaticembryos. The procedure generally includes the steps of (i) germinatingembryos and (ii) inducing the growth of cacao plantlets.

[0060] Somatic embryos may be germinated on a primary embryo conversionmedium. The “primary embryo conversion medium” contains the basal mediumas described for the basal medium of the primary callus growth medium(e.g. DKW medium), and is fortified with KNO₃ in the concentration fromabout 0.1 g/l to 1.0 g/l, and preferably about 0.3 g/l. The medium alsocontains glucose or sucrose as a carbon source. The concentration of thecarbon source may be as generally used in the art, such as for examplefrom about 5 g/l to about 30 g/l. The somatic embryos are germinatedunder light for a period of about 10 to about 30 days at temperaturesknown in the art and for example at 25±5° C.

[0061] To achieve regeneration of whole cacao plantlets, germinatingembryos are transferred onto a secondary embryo conversion medium. The“secondary embryo conversion medium” contains a diluted basal mediumsupplemented with a source of potassium and nitrogen, such as forexample, KNO₃ and a carbon source which may be glucose, sucrose or acombination thereof. The “basal” medium may be as described for theprimary callus growth medium, but other media known in the art such asMS medium may be used. The choice of the basal medium is not critical inthis step. However, the basal medium is “diluted,” to facilitate theautotrophic development of germinating embryo-derived plantlets. Variousdilutions of known media may be used and determining the most useful oneis a matter of optimization. For example a 1:2 to 1:10 dilution may beused.

[0062] The source of potassium and nitrogen can be any compound or acombination of compounds that provide K and NO₃ ions. Preferably, KNO₃is used.

[0063] The carbon source may be used in the concentration generallyknown in the art. For example, when a combination of glucose and sucroseis used, glucose is in the amount from about 1 g/l to about 20 g/l andsucrose is in the amount from about 1 g/l to about 10 g/l. If glucosealone is used, the amount is from about 1 g/l to about 20 g/l. Ifsucrose alone is used, the amount is from about 5 g/l to about 40 g/l.

[0064] According to one embodiment of the invention, the steps ofgerminating embryos and regenerating cacao plantlet described above maybe performed in a single step by transferring somatic embryos to a plantregeneration medium and culturing the embryos until plantlets areformed. The “plant regeneration medium” contains a diluted basal mediumas described above for the secondary conversion medium and issupplemented with a carbon source and a growth hormone. Any auxin may beused in this medium, such as for example, IAA, NAA and IBA. In oneembodiment, gibberellic acid and a combination of glucose and sucrose isused. Gibberellic acid may be used in the range of about 0.01-3.0 mg/l,preferably about 0.05-1.0 mg/l, and most preferably about 0.1-0.3 mg/l.

[0065] The pH of the novel culture media prepared and used according tothe present invention is as generally known in the prior art.Preferably, the pH ranges from about 4.0 to about 6.0 and mostpreferably from about 5.5 to about 5.8. It is within the skill of aperson of skill in the art to optimize the pH of the culture mediumusing the guidance of the present specification and general knowledge inthe art.

[0066] The advantage of the methods of the present invention is that thetotal time required to produce somatic embryos and cacao plantlets isreduced in comparison with the methods known in the art. The amount oflabor and the cost involved are greatly reduced. More significantly, theefficiency of somatic embryo production and plant regeneration fromcacao explants is dramatically improved. In total, these improvementsallow for the practical use of somatic embryogenesis for cacao clonalpropagation and other applications that require the production of alarge quantity of plants from limited source materials.

Agrobacterium-mediated Transformation

[0067] In the last decade, transformation technology has played anincreasingly important role in the genetic manipulation of crop plantsfor their improvement and the study of the molecular mechanismsunderlying plant gene expression and regulation. However, due to thelack of a useable transformation procedure, the application of suchbiotechnological approaches has not been possible for cacao. Successfultransformation of cacao cells, and the subsequent production oftransgenic somatic embryos and plants using the Agrobacterium-mediatedtransformation procedures as described herein, provide a new procedurefor the introduction of foreign genes into cacao and an alternativeapproach for the incorporation of novel mechanisms of resistance toviruses, fungi and insect pests. A “foreign gene” is intended to meanany gene or polynucleotide not naturally found in cacao. In addition,this technology enables the development of transgenic cacao varietieswith improved agronomic performance characteristics, and provides a newexperimental system for study of gene expression and function, in cacao.The use of cacao varieties improved via the utilization oftransformation technology also facilitates the implementation ofsustainable agricultural practices in cacao cultivation, and eventuallyhelps maintain a healthy tropical forest ecosystem.

[0068] Transgenic cacao plants may be produced according to the methodof the invention having the following steps: (i) culturing Agrobacteriumin low-pH induction medium at low temperature and preconditioning, i.e.,coculturing bacteria with wounded tobacco leaf extract in order toinduce a high level of expression of the Agrobacterium vir genes whoseproducts are involved in the T-DNA transfer; (ii) coculturing cacaotissue explants, including zygotic and/or somatic embryo tissues derivedfrom cultured explants, with the incited Agrobacterium; (iii) selectingtransformed callus tissue on a medium containing antibiotics; and (v)and converting the embryos into plantlets.

[0069] Any non-tumorigenic A. tumefaciens strain harboring a disarmed Tiplasmid may be used in the method of the invention. Any Agrobacteriumsystem may be used. For example, Ti plasmid/binary vector system or acointegrative vector system with one Ti plasmid may be used. Also, anymarker gene or polynucleotide conferring the ability to selecttransformed cells, callus, embryos or plants and any other foreign genesuch as for example a gene conferring resistance to a disease may alsobe used. A person of skill in the art can determine which markers andforeign genes are used depending on particular needs. For example, A.tumefaciens strain EHA101 harboring a disarmed version of theatropine-type supervirulent Ti plasmid pTiBo542 (Hood et al. 1986. JBacteriol 168:1291-1301) and a binary vector pDM96.0501 (shown in FIG.11) may be used.

[0070] For purposes of the present invention, “transformed” or“transgenic” means that at least one marker gene or polynucleotideconferring selectable marker properties is introduced into the DNA ofcacao cell, callus, embryo or plant. Additionally, any foreign gene mayalso be introduced.

[0071] To increase the infectivity of the bacteria, Agrobacterium iscultured in low-pH induction medium, i.e., any bacterium culture mediawith a pH value adjusted to from 4.5 to 6.0, most preferably about 5.2,and at low temperature such as for example about 19-30° C., preferablyabout 21-26° C. The conditions of low-pH and low temperature are amongthe well-defined critical factors for inducing virulence activity inAgrobacterium (E.g. Altmorbe et al. (1989) Mol. Plant-Microbe. Interac.2:301-308; Fullner et al. (1996) Science 273:1107-1109; Fullner andNester (1996) J. Bacteriol. 178:1498-1504).

[0072] The bacteria is then preconditioned by coculturing with woundedtobacco leaf extract to induce a high level of expression of theAgrobacterium vir genes. The preconditioning with tobacco extract isknown in the art and is described in detail in Example 3. The vir genesare involved in the T-DNA transfer process as generally known in theart. The wounded tobacco leaf extract is prepared as generally known inthe art.

[0073] Agrobacterium treated as described above is then cocultured withcacao tissue explants, such as for example zygotic and/or somatic embryotissue. Somatic embryos may be obtained according to the presentinvention as described above. Cacao explants are then cultured on aliquid, semi-solid or solid tissue culture medium containing selectiveantibiotics to obtain transformed callus masses. The above-described PCGand SCG media may be used in this process according to the stepsdescribed under “Methods of Inducing Somatic Embryos.” The transformedcallus may be identified based on any selective marker such as forexample expression of the kanamycin-resistance gene (NPTII) and thegreen fluorescent protein (GFP) gene, incorporated in the T-DNA regionof the binary vector.

[0074] In the next step, transgenic somatic embryos are induced from therecovered transformed calli. The above-described embryo developmentmedium and culture procedure may be used.

[0075] In the final steps, embryos are converted and cacao plantlets areregenerated using a combination of a primary and secondary embryoconversion media, a plant regeneration medium described above or anyother medium useful for embryo conversion and regeneration of plantlets.

[0076] The culture media used in the invention contain an effectiveamount of each of the above described medium components (e.g. basalmedium, growth regulator, carbon source). For purposes of the presentinvention, an “effective amount” of a given medium component is theamount necessary to cause a recited effect. For example, an effectiveamount of a growth hormone in the primary callus growth medium is theamount of the growth hormone that induces callus formation when combinedwith other medium components. Other compounds known in the art to beuseful for tissue culture media, such as for example vitamins andgelling agents, may also be used as optional components of the culturemedia of the invention.

[0077] The present invention also relates to cacao somatic embryos andplants, as well as transformed somatic embryos and transformed plantsobtained using the methods described herein.

[0078] The invention is further described by the following non-limitingexamples.

EXAMPLE 1 Materials and Methods

[0079] Chemicals from Sigma Chemical Co., St. Louis, Mo. were used forall media preparation. Calcium hypochlorite [Ca(OCl)₂] was obtained fromAldrich Chemical Company, Inc. (Milwaukee, Wis.). The pH of the mediumwas adjusted using 1N KOH, prior to autoclaving. All media wereautoclaved for 20 min at 121° C.

[0080] A powdered form of the DKW medium developed by Driver andKuniyuki (Driver, J. A. and Kuniquki, A. H., (1984) Hortsci 19: 507-509)and Tulecke and McGranahan (Tulecke, W. and McGranaham, G., (1985) L.Plant Sci 40: 57-63) was obtained from Sigma Chemical Co. (D-6162).However, due to the hygroscopic nature of the powdered preparation,stock solutions containing the chemical components of the DKW mediumwere used for medium preparation. Macronutrients of the DKW medium wereseparated into stock solutions A and B to avoid chemical interactionsbetween inorganic salts at high concentrations, and to preventprecipitation of salts during storage.

[0081] DKW 10×macro solution A was prepared by combining (per liter)14.16 g NH₄NO₃ and 19.68 g Ca(NO₃)₂.4H₂O. DKW 10×macro solution B wasprepared by combining the following compounds (per liter): 1.49 gCaCl₂.2H₂O, 15.59 g K₂SO₄, 7.4 g MgSO₄.7H₂O, and 2.65 g KH₂PO₄. DKW100×micro solution was prepared by combining the following compounds(per liter): 1.7 g Zn(NO₃)₂.6H₂O, 3.34 g MnSO₄.H₂O, 3.38 g FeSO₄.7H₂O,9.54 g Na-EDTA, 0.48 g H₃BO₃, 25mg CuSO₄.5H₂O, and 39 mg Na₂MoO₄.2H₂O.DKW 100×vitamin solution was prepared by combining the followingcompounds (per liter): 10 g myo-inositol, 0.2 g thiamin-HCl, 0.1 gnicotinic acid, and 0.02 g glycine.

[0082] Fresh stock solutions of growth regulators (e.g. TDZ and 2,4-D),were prepared every 3 months. TDZ solution was prepared by dissolving 5mg thidiazuron in 1 ml of 1N KOH and dH₂O. 2-4-D solution was preparedby dissolving 10 mg 2,4-dichlorophenoxyacetic acid in 1 ml 100% ethanoland adding dH₂O. Kinetin solution was prepared by dissolving 10 mgkinetin in 1 ml of 1N NaOH and adding dH₂O. 6-BA solution was preparedby dissolving 10 mg 6-benzylaminopurine in 1 ml of 1N NaOH and addingdeionized water.

[0083] Primary callus growth (PCG) medium was prepared by combining thefollowing solutions and compounds (per liter): 100 ml each DKW macrosolutions A and B, 10 ml DKW micro solution, 1 ml DKW vitamin solution,20 g glucose, 250 mg glutamine, 100 mg myo-inositol, 100 ml 2,4-Dsolution, 10 ml TDZ solution, and 2.0 g phytagel, and the pH wasadjusted to 5.8.

[0084] Secondary callus growth (SCG) medium was prepared by combiningthe following solutions and compounds: 2.3 g McCown's woody plant basalsalt mixture (Lloyd, D. and McCown, B., (1981) Proc Int Plant Prop Soc30: 421-427) (available from Sigma M-6774). 1.0 ml Gamborg's vitaminsolution (Gamborg, O. L., (1966) Can J Biochem 44: 791-799) (availablefrom Sigma G-1019), 20.0 g glucose, 200 μl 2,4-D solution (=2.0 mg/l),30 μl kinetin solution (=0.3 mg/l), 50 ml coconut water, and 2.2 gphytagel, and the pH was adjusted to 5.8.

[0085] Embryo development (ED) medium was prepared by combining thefollowing solutions and compounds (per liter): 100 ml each DKW macrosolutions A and B, 10 ml DKW micro solution, 1 ml DKW vitamin solution,20 g sucrose, 1.0 g glucose, and 2.0 g phytagel, and the pH was adjustedto 5.8. Autoclaved ED medium often solidified quickly at a relativelyhigh temperature (lower than 40° C.), possibly due to its high contentof calcium salts that may trigger chemical reactions with phytagel.Thus, precaution must be taken during distribution of the autoclavedmedium into culture plates, in order to prevent over-cooling andpremature solidification of the medium.

[0086] MSG medium was prepared by combining the following solutions andcompounds (per liter): 4.44 g MS basal salts (Murashige, T. and Skoog,F., (1962) Physiol Plant 15:473-497) with Gamborg's vitamins (Gamborg,O. L., (1966) Can J Bioichem 44:791-799), 20 g glucose, and 2 gphytagel, and the pH was adjusted to 5.8.

[0087] Plant regeneration (PR) medium was prepared by combining thefollowing solutions and compounds (per liter): 20 ml each DKW macrosolutions A and B, 2.0 ml DKW micro solution, 0.2 ml DKW vitaminsolution, 10 g glucose, 5 g sucrose, 0.2 g KNO₃, and 1.7g phytagel, andthe pH was adjusted to 5.8.

[0088] Plant materials used were unopened immature cacao flower buds 5to 8 mm in length (depending upon genotype), collected between 8 am and11 am. Flower buds at advanced developmental stages, harvested in theafternoon may also be used. However, these buds open readily duringsurface-sterilization and may cause the contamination of explants.Flower buds collected during the morning remain closed throughout thesurface-sterilization process, and are the preferred explants.

Experimental Procedure

[0089] Staminode and petal base tissues were used as culture explants(FIG. 2). Although immature flower buds with a range of sizes can becollected, large flower buds were chosen because such flower buds wereeasier to dissect and handle in the absence of a dissecting microscope.In addition, staminodes and petal base explants were separated fromassociated floral parts such as stamen filaments and petal tissue, inorder to minimize possible interactions that may affect the in vitrogrowth of explants. It was found that stamen-derived calli weredifficult to induce to produce somatic embryos, and that petal tissuesturn brown quickly and released phytotoxic phenolic compounds into themedium.

[0090] a. Collection and Surface-sterilization of Flower Buds

[0091] Immature flower buds were collected in a 50-ml centrifuge tubecontaining cold water. A 1% (w/v) calcium hypochlorite solution wasprepared by dissolving 0.5 g Ca(OCl)₂ in 50 ml sterile water in asterile 50-ml centrifuge tube. The cold water was decanted from thecentrifuge tube containing the immature flower buds inside the transferhood and all of the flower buds were transferred into the sterilecentrifuge tube containing the calcium hypochlorite solution. The flowerbuds were immersed in the calcium hypochlorite solution for 20 min. Thehypochlorite solution was then removed and 40 ml sterile water was addedto rinse the flower buds. The buds were rinsed at least three times andwere then transferred to a Petri dish and the plate was covered toprevent desiccation.

[0092] b. Dissection of Flower Bud and Callus Induction

[0093] Two to three layers of sterile paper towels were placed in thetransfer hood. Four flower buds on the top surface of the paper towelswere blotted dry and then transferred onto a Petri dish cover. Theflower buds were sliced across at a position of about ⅓ of the flowerlength from the base using a sterile scalpel blade. The staminodes andpetal base tissues were extracted together from the top part of theflower bud using a pair of sterile forceps. Any attached petal tissuewas removed from the petal base explants.

[0094] The staminodes and petal base explants from the four flower budswere then transferred into a Petri dish containing 30 ml of PCG medium.Any fused staminodes and petal base explants were separated and explantswere distributed evenly across the medium. The Petri dishes were sealedwith a double layer of parafilm and cultures were maintained in the darkat 25±2° C. for 14 days (FIG. 2a). Growth reduction, senescence, andtissue browning may occur with subculture intervals longer than 14 days.

[0095] The staminode and petal base explants were then transferred froma PCG medium to a Petri dish containing 30 ml of SCG medium. The disheswere sealed and the cultures were maintained in the dark for 14 days at25±2° C. Globular calli were produced on the entire tissue explant atthe end of this culture period (FIG. 2b).

[0096] c. Somatic Embryo Induction and Maintenance

[0097] Staminode and petal base explants and calli derived in the callusinduction step were transferred to Petri dishes containing 30 ml of EDmedium. Explants were cultured in the dark for 14 days at 25±2° C.Explants were then subcultured onto the fresh ED medium and maintainedin the dark for another 14 days. By that time, numerous somatic embryosat globular and heart-shaped stages of development were visible on theembryogenic calli (FIG. 2c and d).

[0098] Somatic embryos were then excised from the callus tissue andtransferred onto Petri dishes containing 30 ml of ED medium. Embryocultures were maintained in the dark with a subculture interval of 14days at 25±2° C., until somatic embryos reached maturity (FIG. 2e).

[0099] d. Embryo Conversion and Plant Establishment

[0100] Germinating somatic embryos with an extended radicle, preferablyType II embryos (FIG. 2 f) were selected. The embryos were insertedvertically into PR medium in a Magenta vessel (80 ml/vessel). Four tofive embryos were placed in each vessel. The vessel was sealed withlow-temperature electric tape. Cultures were maintained under light(16-h photoperiod) at 25±2° C. for 14 days. Germinating embryos weresubcultured to fresh PR medium every 14 days.

[0101] For Type I embryos, mature embryos about 1 cm in length, wereselected and transferred onto Petri dishes containing 30 ml MSG medium.Cultures were maintained under light with a 24-h photoperiod for 14days. The embryos with roots and shoots that turned green weretransferred onto PR medium in Magenta vessels (FIG. 2g). Cultures weremaintained under light (16-h photoperiod) at 25±2° C. for 14 days.Germinating embryos were subcultured to fresh PR medium every 14 days.

[0102] Plantlets with developing green leaves and healthy taproots weretransferred into 4-inch plastic pots containing sterile Metro-Mix 300soil mixture (FIG. 2h). Water was poured into the pot to saturate thesoil mixture. The plantlet was covered using a magenta vessel. Plantswere maintained in the greenhouse with an 80% humidity controlled by anautomatic misting system. Water was added regularly to maintain anadequate moisture content for optimal plant growth. When the plantletproduced a new leaf, the cover vessel was removed. Regular amounts offertilizers were applied to enhance plant growth (FIG. 2h).

Experimental Results

[0103] A key to the successful development of a highly efficient somaticembryogenesis system for cacao was the discovery and use of a series ofimproved culture conditions throughout the entire procedure. In allprevious studies of somatic embryogenesis in cacao, the MS medium(Murashige and Skoog, (1962) Physiol Plant 15:473-497) that waspreviously developed for the in vitro culture of tobacco tissue, wasemployed as the main source of inorganic nutrients for cacao cellgrowth. However, the use of this medium was in fact one of the majorlimitations in cacao tissue culture. The DKW medium, which wasformulated for somatic embryogenesis and plant regeneration in woodyperennials, provided a better balanced composition of nutrients forcacao. In this experiment, the use of DKW medium was essential forobtaining embryogenic cultures and for maintaining normal growth anddevelopment of cacao somatic embryos.

[0104] High frequencies of somatic embryo production and plantregeneration from cultured floral explants were readily obtained. Aschematic outline of cacao somatic embryogenesis used in this experimentis shown in FIG. 1. Photographs which show efficient somaticembryogenesis and plant regeneration from cultured staminode tissues ofcacao through depiction of the various products obtained throughout thestages of the procedure are shown in FIG. 2.

[0105] The effect of TDZ concentration on somatic embryogenesis fromstaminode explants of five cacao genotypes was determined and is shownin FIG. 3. Staminodes were cultured on PCG medium containing variousconcentrations of TDZ. Embryonic calli were subcultured onto SCG mediumand somatic embryos were subsequently induced by culturing calli on EDmedium. Data were collected two months after culture initiation. Eachtreatment contained 20 staminodes per plate with three replicate plates.The values shown in FIG. 3 represent the average percentage rates ofembryo-producing staminodes from three repeated experiments. Up to 100%of cultured staminode and over 60% of petal base explants from cacaogenotype Sca-6 produced somatic embryos (FIG. 3).

[0106] The effect of TDZ concentration on somatic embryo production fromstaminode explants of five cacao genotypes was determined and is shownin FIG. 4. Staminodes were cultured on PCG medium containing variousconcentrations of TDZ. Embryogenic calli were subcultured onto SCGmedium and somatic embryos were subsequently induced by culturing callion ED medium. Data were collected two months after culture initiation.Each treatment contained 20 staminodes per plate with three replicaplates. Values represent the average number of somatic embryos perresponsive staminode from three repeated experiments. A single Sca-6staminode explant produced up to 140 and an average of about 46 primarysomatic embryos (FIG. 4).

[0107] The level of somatic embryo formation from staminode explants of19 tested cacao genotypes was determined and is shown in FIG. 5.Embryogenic calli were induced and propagated on PCG and SCG media,respectively. Somatic embryos were induced by culturing calli on EDmedium. Data were collected two months after initiation. Each treatmentcontained 20 staminodes per plate with three replicate plates. Valuesrepresent the average percentage rates of embryo-producing staminodesfrom two to three repeated experiments.

[0108] The average number of somatic embryos produced from responsivestaminode explants of 19 tested cacao genotypes was determined and isshown in FIG. 6. Embryogenic calli were induced from staminode explantson PCG medium for 14 days and propagated on SCG medium for another 14days. Somatic embryos were subsequently induced by culturingstaminode-derived calli on ED medium. The number of somatic embryos fromeach responsive staminode explant was determined two months afterculture initiation. Each treatment contained 20 staminodes per platewith three replicate plates. Values represent the average number ofsomatic embryos per responsive staminode from two to three repeatedexperiments.

[0109] The results of FIGS. 5 and 6 indicate that the success of theprocedure was genotype-independent. All 19 tested cacao genotypesproduced somatic embryos.

[0110] Important factors in the invention included the use of TDZ andglucose as sources of cytokinin and carbon, respectively for theinitiation of highly embryogenic cultures of cacao. TDZ possesses astrong cytokinin-like activity exceeding most of other commonly usedcytokinins including zeatin, BA, and kinetin, and is highly resistant todegradation by cytokinin oxidase. However, over the years TDZ had notbeen tested for somatic embryogenesis in cacao. The invention devisedfor the first time the proper means to utilize this potent and stablecompound for effective induction of embryogenic callus from cacao floralexplants and subsequent efficient production of somatic embryos (FIG. 3and 5). In all previous studies of cacao somatic embryogenesis, sucroseand maltose were used as the main carbon source. The use of glucose as amajor carbon source in the invention resulted from the observation thatcacao tissues cultured on glucose-containing medium grew normally anddid not produce any hypersensitive reactions that often lead to tissuesenescence and cell death as frequently observed in cultures using othersugars.

[0111] Two types of somatic embryos were identified based on thefollowing characteristics: Type I embryos had a yellowish and vitrifiedappearance, and an expanded embryo axis. During extended culture on EDmedium, mature Type I embryos tended to remain dormant. After transferto embryo conversion medium, these embryos showed extensive cotyledonarygrowth, followed by the development of true leaves. Root development ingerminating Type I embryos was normally slow. Type II somatic embryoswere whitish in color and had a defined embryonic axis structure. Theseembryos underwent spontaneous germination upon reaching maturity on EDmedium. After transfer to embryo conversion medium, these embryos turnedgreen quickly, exhibited a significant hypocotyl elongation, andproduced a strong taproot, within a short period of time. Epicotylelongation and production of true leaves often occurred 2 to 3 weeksafter transfer. The plant regeneration responses of these two types ofcacao somatic embryos using previously defined culture conditions aresummarized in Table 3 which shows data in which mature somatic embryosof cacao genotype Sca-6 were cultured on PR medium in Magenta vessels.Data were collected two months after culture initiation. Up to 73% ofthe selected mature somatic embryos that were produced using thisprocedure were capable of conversion into plantlets (Table 3). TABLE 3Plant Regeneration Response of Two Types of Somatic Embryos of CacaoEmbryo Type Type I % Type II % Total No. of SE 96 — 191 — SE with root31 32.3 183 95.8 SE with shoot 26 27.1 140 73.3

EXAMPLE 2 Materials and Methods

[0112] Materials and methods were as described in Example 1 with thefollowing exceptions.

[0113] In the secondary callus growth medium, kinetin and coconut waterwere replaced by 5 μl of 6-BA stock solution (=0.05 mg/l).

[0114] Primary embryo conversion (PEC) medium was prepared by combiningthe following solutions and compounds (per liter): 100 ml DKW macrosolutions A and B, 10 ml DKW micro solution, 1 ml DKW vitamin solution,0.3 g KNO₃, 1 ml amino acid solution, 20 g glucose, and 1.75 g phytagel,and the pH was adjusted to 5.8. Amino acid 100×solution for use in theprimary embryo conversion medium was prepared by combining the followingcompounds (per 100 ml ): 43.55 mg arginine, 18.76 mg glycine, 32.8 mgleucine, 45.65 mg lysine, and 51.05 mg tryptophane.

[0115] Secondary embryo conversion (SEC) medium was prepared bycombining the following solutions and compounds (per liter): 25 ml eachDKW macro solutions A and B, 2.5 ml DKW micro solution, 0.25 ml DKWvitamin solution, 5.0 g glucose, 2.5 g sucrose, 0.2 g KNO₃, and 1.75 gphytagel, and the pH was adjusted to 5.8.

Experimental Procedure

[0116] Steps a, b, and c were performed as described in Example 1. Stepd was as described below.

[0117] d. Embryo Conversion and Plant Establishment

[0118] Mature somatic embryos (generally up to 2 cm in length) withdistinctive cotyledons or with an extended radicle (about 0.5 cm inlength), including both Type I and Type II embryos (FIG. 2f) wereselected. Ten to fifteen embryos were placed horizontally on PEC medium,in the Petri dishes. The culture dishes were sealed with a double layerof parafilm and maintained under light (16-h photoperiod) at 25±2° C.for 20 days. Germinating embryos were subcultured to fresh PEC mediumevery 20 days until the emergence of shoot growth was observed.

[0119] Shoot-producing embryos with two green leaves of at least 1 cm inlength were transferred to SEC medium in Magenta vessels. The embryoswere placed horizontally on the surface of the medium at a density of 4to 6 embryos per vessel. Culture vessels were sealed with a double layerof parafilm. Cultures were maintained under light with a 16-hphotoperiod for 30 days. Embryos that produced no roots or weak rootsshorter than about 2 cm were transferred onto fresh SEC medium.Plantlets with roots longer than 2 cm were left in the culture vessel toavoid breakage of the delicate root system due to transfer handling.These plantlets can be maintained in the same culture vessel for up to 4months without any adverse effects on plant growth and development (FIG.2g).

[0120] Plantlets with developing green leaves of more than 3 cm inlength and healthy roots of more than 2 cm in length were transplantedinto 4-inch plastic pots containing autoclaved pre-moistened Metro-Mix500 soil mixture (FIG. 2h). Water was poured into the pot to saturatethe soil mixture. Plantlets were maintained in the greenhouse with 90%humidity controlled by an automatic misting system. Water was addedregularly to maintain an adequate moisture content for optimal plantgrowth. When the plantlet produced a new leaf, regular amounts offertilizers were applied to enhance plant growth (FIG. 2h).

Experimental Results

[0121] The influence of the concentration of 6-benzylaminopurine (6-BA)used in the SCG medium on the frequency of somatic embryo productionfrom cacao staminodes was determined and is shown in FIG. 7. Rapidlygrowing calli were initiated from staminode explants on PCG medium andthen transferred onto SCG medium containing various concentrations of6-BA. A control treatment using SCG medium containing kinetin andcoconut water (Kn/cm) was included. Somatic embryos were induced byculturing embryonic calli on ED medium. Data were collected two monthsafter culture initiation. Each treatment contained 20 staminodes perculture plate with three replicate plates per experiment. Bar valuesrepresent the average percentage rates of embryo-producing staminodesfrom two independent experiments.

[0122] The influence of the concentration of 6-BA used in the SCG mediumon the production of somatic embryos from cacao staminode explants ofSca-6 was determined and is shown in FIG. 8. Rapidly growing calli wereinitiated on PCG medium and then transferred onto SCG medium containingvarious concentrations of 6-BA. A control treatment using SCG mediumcontaining kinetin and coconut water (Kn/cm) was included. Somaticembryos were induced by culturing embryogenic calli on ED medium. Datawere collected two months after culture initiation. Each treatmentcontained 20 staminodes per culture plate with three replicate platesper experiment. Bar values represent the average number of somaticembryos produced from each embryo-producing staminode from twoindependent experiments.

[0123] In addition, efficient repetitive embryogenesis was alsoobtained. A single primary embryo produced more than 50 secondaryembryos after being subjected to an extended culture period of one month(data not shown).

EXAMPLE 3

[0124] The following example describes an applications of theabove-described Agrobacterium-mediated transformation of cacao using A.tumefaciens strain EHA101 harboring a disarmed version of theatropine-type supervirulent Ti plasmid pTiBo542 (Hood et al. 1986. JBacteriol 168:1291-1301) and a binary vector pDM96.0501 andstaminode-derived somatic embryos as inoculation explants.

Materials and Methods

[0125] A binary plasmid, pDM96.050, 1, which contains three transgenes(SGFP-TYG, GUS and NPTI1 genes, all under control of CaMV35S promoter)within the T-DNA region and a gentamycin-resistance gene in the plasmidbackbone (FIG. 3) was obtained from Calgene Inc., Davis, Calif. Theplasmid was introduced into A. tumefaciens strain EHA101 for use incacao transformation.

[0126] Chemicals from Sigma Chemical Co., St. Louis, Mo., FisherScientific, Pittsburgh, Pa., and Difco Laboratories, Detroit, Mich. wereused for all media preparation. The pH of the media was adjusted using1N KOH solution, prior to autoclaving. All media were autoclaved for 20min at 121° C.

[0127] The preparation of DKW stock solutions and embryo development(ED) medium was as described in Example 1.

[0128] YEP5 medium was prepared by combining 10.0 g peptone, 10.0 gyeast extract, and 5.0 g NaCl. The pH was adjusted to 5.2.

[0129] Agrobacterium inoculation (AI) medium was prepared (per liter) bycombining 0.88 g MS basal salts plus Gamborg's vitamins (Sigma mediumpreparation, Cat. No. M-0404) and 10 g glucose. The pH was adjusted to5.3.

[0130] Primary selection (PS) medium was prepared (per liter) bycombining ED medium with 400 mg Claforan (i.e., cefotaxime sodiumavailable from Hoechst-Russel Pharmaceutical Inc., Somerville, N.J.).

[0131] Secondary selection (SS) medium was prepared (per liter) bycombining ED medium without phytagel, 400 mg Claforan, and 200 mgkanamycin.

[0132] Tobacco propagation (TP) medium was prepared (per liter) bycombining 4.4 g MS basal salts plus Gamborg's vitamins, 30.0 g sucrose,and 2.2 g phytagel. The pH was adjusted to 5.8.

[0133] Plant materials used were: In vitro grown tobacco (Nicotianatabacum cv. Xanthi) and Cacao somatic embryos at heart- andtorpedo-shaped stages of development.

Experimental Procedure

[0134] a. Preparation of Bacterial Culture and Inoculation of CacaoSomatic Embryos

[0135] Prior to inoculation of cacao somatic embryos, Agrobacteriumcells were treated with tobacco extract prepared from wounded leaftissues of in vitro grown tobacco plants. To achieve optimal stimulationof the expression of Agrobacterium vir genes by wound-inducedmetabolites and other cellular factors, tobacco leaves were wounded andpre-cultured overnight. Culturing of bacteria in low pH medium and atlow temperature was used to further enhance the bacteria vir geneexpression and infectivity.

[0136] Cacao somatic embryos were produced following the proceduresdescribed in Examples 1 and 2. Healthy whole embryos of about 4 to 8 mmin size, at heart- and torpedo-shaped developmental stages were used fortransformation. In order to reduce the phytotoxic effects to embryogrowth, any embryos with brown spots containing phenolics compounds ordying tissues were eliminated from the transformation materials.

[0137] Twenty-four hours before the transformation experiment three tofive leaves were removed from in vitro grown tobacco plants andcollected in a Petri dish. In vitro tobacco plants were maintained inMagenta vessels containing 80 ml of TP medium at 25° C. with a lightingregime of 16 hours and a light intensity of about 40 mmol ×m-2×s-1.Tobacco plants were subcultured every 20 days by cutting andtransferring shoot tips with 2 to 3 expanded leaves to fresh medium. Thetobacco leaves were cut into small pieces (0.5 cm²). Leaf pieces werespread evenly across the surface of a Petri dish containing 25 ml of TPmedium. A total of 5 g of leaf tissue was prepared in this manner. ThePetri dishes were sealed and cultured overnight in the dark.

[0138] One ml of A. tumefaciens stock solution was inoculated into 30 mlof YEP5 medium supplemented with 50 mg/l kanamycin and 20 mg/lgentamycin. Bacteria were cultured overnight at 25° C. in the dark on agyratory shaker at 100 rpm. When the bacteria density reached an OD₆₀₀of 1.0 to 1.5, the cells were harvested by centrifugation at 7000 rpm at20° C. for 10 min. The supernatant was discarded and the bacteria wereresuspended in 20 ml AI medium in a sterile flask. Five grams ofpre-cultured tobacco leaf tissue was transferred to a sterile mortar.Two ml of sterile water was added. Leaf tissue was ground into a fineslurry. The aqueous solution (about 5 ml ) was transferred from theslurry to the flask containing the Agrobacterium using a steriletransfer pipet. The bacteria mixture was cultured at 22° C. in the darkon a shaker at 100 rpm for 3 hours.

[0139] About 100 healthy intact embryos were selected from somaticembryo cultures prepared according to the procedures of Examples 1 and2. Unlike the common practices used in Agrobacterium-mediatedtransformation of other plant species, it is not necessary to wound thecacao somatic embryos before Agrobacterium infection. Wounding embryotissue induces the production of phenolic compounds and results inreduced infection rate.

[0140] The developmental stages of cacao somatic embryos can influencethe transformation efficacy. According to the applicants' transient geneexpression experiments, the use of heart-shaped embryos resulted in thehighest transformation frequency, followed by torpedo-shaped embryos,while the globular embryos tended to die easily, probably due to thesmaller tissue size and lower tolerance to physical wounding and stresscaused by the Agrobacterium infection (FIG. 4). Thus, the use of heart-and torpedo-shaped embryos for transformation is preferred. Embryos weretransferred to a Petri dish. About 30 ml of pretreated Agrobacteriumculture was poured into the Petri dish. Forceps were used to ensureimmersion of all explants into the suspension. The Petri dish wascovered and placed in a vacuum desiccator. Vacuum pressure was appliedfor 1 min. The vacuum was slowly released. The liquid solution wasremoved completely using a sterile transfer pipet. The embryos wereplaced on the surface of a piece of sterile paper towel for a fewseconds to remove the remaining solution. The embryos were transferredto Petri dishes containing 30 ml of ED medium (30 embryos per plate).The Petri dishes were covered and sealed. The Petri dishes wereincubated at 25° C. in the dark for 24 hours.

[0141] b. Detection of GFP Expression and Selection for Transformants

[0142] GFP was used to monitor the transient expression of transgenes incacao cells soon after Agrobacterium-mediated transformation and tofacilitate the identification of explants containing transformed cellsfor subsequent selection for stable transformants using liquid culturemedium.

[0143] After co-cultivation with Agrobacterium on ED medium, all of theinoculated embryos were transferred into a sterile 50-ml centrifuge tubecontaining 40 ml of sterile water. The tube was inverted several timesto wash out attached bacteria from the surface of the embryos. Thesupernatant was removed completely. Forty ml of sterile water containing400 mg/l Claforan was added to the tube. The washing process wasrepeated and the aqueous solution was removed. The embryos were placedbriefly on the surface of a sterile paper towel to remove excessivewater. The embryos were transferred onto a Petri dish containing 30 mlof PS medium. The dishes were sealed and cultured at 25° C. in the darkfor 4 days. During this period of primary selection on solid medium, thepresence of green fluorescence derived from the expression of GFP geneon the surface of inoculated embryos was monitored on a daily basisusing a dissecting microscope equipped with an MVI fluorescenceattachment. Tissues were illuminated with blue light and GFP emissionwas monitored using green baud pass FITC filters. The position ofGFP-positive embryos was marked on the culture plate. Based on thetransient gene expression studies, the GFP expression in inoculatedcacao embryos became detectable 4 days after Agrobacterium infection(FIG. 13). Since then, the GFP expression frequency gradually increased,up to the 8th day, and then started to decline. Accordingly, inoculatedembryos can be monitored for GFP expression for more than 6 days afterinfection, provided that the bacteria do not overgrow on the surface ofthe embryo tissue. In this way, more GFP-positive embryos may beidentified and used in the subsequent selection process.

[0144] About 20 GFP-positive embryos were transferred into a sterileflask containing 30 ml of SS medium. The flasks were covered and sealedwith parafilm. Cultures were maintained on a gyratory shaker at 100 rpmunder dim light. Embryos were subcultured to fresh medium every 10 days.During this selection period, the cell division activity of theGFP-expressing foci was inspected regularly to identify the formation ofGFP-positive microcalli on the surface of inoculated embryos.GFP-positive microcalli were excised and transferred to Petri dishescontaining 30 ml of SS medium solidified with 2.0 g/l phytagel. Thecalli were cultured in the dark at 25±2° C. with a subculture intervalof 14 days.

[0145] GFP-positive calli about 3 mm in size were transferred andcultured on solid SS medium modified to contain 100 mg/l Claforan and 50mg/l kanamycin. Secondary embryos produced from the transformedGFP-positive callus tissue were transferred to Petri dishes containing30 ml of ED medium. Transgenic plantlets were recovered by following theculture steps for somatic embryo maturation and plant regenerationdescribed in Examples 1 and 2.

Experimental Results

[0146] The procedure used in this example for Agrobacterium-mediatedtransformation of cacao somatic embryos is outlined schematically inFIG. 9. The expression of GFP in putative transgenic somatic embryos ofcacao after Agrobacterium-mediated transformation was measured and isshown in FIG. 10.

[0147] The effects of developmental stages of cacao somatic embryos andgene promoters on transient GFP expression after Agrobacterium-mediatedtransformation were determined and are shown in FIG. 12. Somatic embryosof cacao genotype Sca-6 at various developmental stages were inoculatedwith A. tumefaciens strain EH101 carrying binary plasmids of eitherpDM96.0501 or pDU96.4451. Inoculated embryos were cultured on ED mediumcontaining Claforan and monitored for GFP expression. Data werecollected 10 days after infection. The values shown in FIG. 12 representthe percentile of globular-, heart-, and torpedo-shaped embryo explantswith either the CaMV35S/GFP or Ubi3/GFP promoter which showed GFPactivity.

[0148] The transient GFP expression in somatic embryos of cacao genotypeSca-6 after Agrobacterium-mediated transformation was monitored and theresults are shown in FIG. 13. Somatic embryos at the heart-shapeddevelopmental stage were infected with pretreated A. tumefaciens strainEHA101 carrying a binary plasmid pDM96.0501 and subsequently subjectedto culture on ED medium containing Claforan. GFP expression ininoculated embryos was monitored daily. Data were averaged from threereplica plates, each containing about 40 embryos. Bar values representthe percentile of embryo explants which showed GFP activity 2, 4, 6, 8,or 18 days after inoculation.

What is claimed is:
 1. A method of inducing somatic embryogenesis in acacao tissue comprising the steps of: (a) providing a cacao tissueexplant; (b) culturing said explant on a primary callus growth medium,said medium having the property of inducing callus growth on saidexplants; (c) culturing the callus produced in step (b) on a secondarycallus growth medium, said medium having the property of inducinghomeostatic growth and bipolar callus cell development; and (d)culturing the callus produced in step (c) on an embryo developmentmedium, said medium having the property of inducing embryodifferentiation.
 2. The method of claim 1 wherein the primary callusgrowth medium comprises a basal medium, a carbon source, and a growthregulator.
 3. The method according to claim 2 wherein said basal mediumis a DKW basal medium.
 4. The method of claim 2 wherein said carbonsource is glucose, sucrose or a combination thereof.
 5. The method ofclaim 2 wherein such growth regulator is thidiazuron,2,4-dichlorophenoxyacetic acid or a combination thereof.
 6. The methodof claim 1 wherein said secondary callus growth medium comprises a basalmedium having a low salt concentration, a carbon source, and a growthregulator.
 7. The method of claim 6 wherein said basal medium is a WPMlow salt basal medium.
 8. The method of claim 6 wherein said carbonsource is glucose, sucrose, or a combination thereof.
 9. The method ofclaim 6 wherein said growth regulator is kinetin, 6-benzyladenine,2,4-D, or any combination thereof.
 10. The method of claim 1 whereinsaid embryo development medium comprises a basal medium and a carbonsource.
 11. The method of claim 10 where said basal medium is a DKWbasal medium.
 12. The method of claim 10 wherein said carbon source issucrose.
 13. The method of claim 1 wherein said explant is a staminodeor a petal base explant.
 14. A method for regenerating cacao plantletscomprising the steps of: (a) providing a cacao somatic embryo; (b)germinating said somatic embryo on a primary embryo conversion medium;and (c) regenerating cacao plantlets on a secondary embryo conversionmedium from germinated embryos produced in step (b).
 15. The method ofclaim 14 wherein said primary embryo conversion medium comprises a basalmedium, a source of potassium and nitrate ions, and a carbon source. 16.The method of claim 15 wherein said basal medium is a DKW basal medium.17. The method claim 15 wherein said carbon source is glucose, sucroseor a combination thereof.
 18. The method of claim 14 wherein saidsecondary embryo conversion medium comprises a diluted basal medium anda carbon source.
 19. The method of claim 18 wherein said carbon sourceis sucrose, glucose or a combination thereof.
 20. The method of claim 14wherein said cacao somatic embryo is produced according to claim 1 . 21.A method of regenerating cacao plantlets comprising the steps of: (a)providing a cacao somatic embryo; (b) culturing said somatic embryo on aplant regeneration medium.
 22. The method of claim 21 wherein said plantregeneration medium comprises a diluted basal medium, a carbon sourceand a growth regulator.
 23. The method of claim 22 wherein said dilutedbasal medium is a WPM low salt basal medium.
 24. The method of claim 22wherein said carbon source is glucose, sucrose or a combination thereof.25. The method of claim 22 wherein said growth hormone is a gibberellicacid.
 26. The method of claim 21 wherein said cacao somatic embryo isproduced according to claim 1 .
 27. A primary callus growth mediumcomprising a basal medium, a carbon source, and a growth regulator, saidmedium having the property of inducing callus growth on cacao tissueexplants.
 28. The medium of claim 27 wherein said basal medium is a DKWbasal medium.
 29. The medium of claim 28 wherein said carbon source isglucose, sucrose or a combination thereof.
 30. The medium of claim 28wherein said growth regulator is thidiazuron, 2,4-dichlorophenoxyaceticacid or a combination thereof.
 31. A secondary callus growth mediumcomprising a basal medium having a low salt concentration, a carbonsource, and a growth regulator, said medium having the property ofinducing homeostatic growth and bipolar cacao callus cell development.32. The medium of claim 31 wherein said basal medium is a WPM low saltbasal medium.
 33. The medium of claim 31 wherein said carbon source isglucose, sucrose, or a combination thereof.
 34. The medium of claim 31wherein said growth regulator is kinetin, 6-benzyladenine, 2,4-D, or anycombination thereof.
 35. An embryo development medium comprising a basalmedium and a carbon source, said medium having the property of inducingembryo differentiation.
 36. The medium of claim 35 wherein said basalmedium is a DKW basal medium.
 37. The medium of claim 35 wherein saidcarbon source is sucrose.
 38. A primary embryo conversion mediumcomprising a basal medium, a source of potassium and nitrate ions, and acarbon source.
 39. The medium of claim 38 wherein said basal medium is aDKW basal medium.
 40. The medium of claim 38 wherein said carbon sourceis glucose, sucrose or a combination thereof.
 41. A secondary embryoconversion medium comprising a diluted basal medium and a carbon source.42. The medium of claim 41 wherein said carbon source is sucrose,glucose or a combination thereof.
 43. A plant regeneration mediumcomprising a diluted basal medium, a carbon source and a growthregulator, said medium having the property of inducing regeneration ofcacao plantlets from cacao somatic embryos.
 44. The medium of claim 43wherein said diluted basal medium is a WPM low salt basal medium. 45.The medium of claim 43 wherein said carbon source is glucose, sucrose ora combination thereof.
 46. The medium of claim 43 wherein said growthhormone is a gibberellic acid.
 47. A cacao somatic embryo producedaccording to claim 1 .
 48. A cacao plant produced according to claim
 14. 49. A cacao plant produced according to claim 20 .
 50. A cacao plantproduced according to claim 21 .